Ultrahyb hybridization buffer

SPINT1 RNA (hg38, chr15:40847819–40848367) was prepared using HiScribe™ T7 High Yield RNA Synthesis Kit (NEB) followed by DNAse I RNAse free (NEB) treatment, purified by phenol chloroform extraction and ethanol precipitation. 50 ng of recombinant Ago2 or Ago3 protein (Active Motif) were pre-incubated with 500 nM single-stranded tsRNA in cleavage buffer (HEPES–KOH 25 mM pH 7.5; MgCl₂ 5 mM; KCl 50 mM; DTT 0.5 mM; EDTA 0.2 mM; ATP 0.1 mM; GTP 0.02 mM; BSA 0.05 μg/μl; Ribolock 20 U; protease inhibitors EDTA free 1×) for 2 h at 37°C and followed by adding 400 ng of spint1 RNA. Cleavage reactions were quenched by TBE–urea loading buffer after 0, 3, 6 and 24 h incubation at 37°C, respectively. Cleavage products were resolved by 4% polyacrylamide 7 M urea denaturing gel at 400 V in 1× TBE buffer. For blotting cleavage products were transferred onto Hybond N+ positive charged nylon membranes (GE Healthcare) by semidry electroblotting in 0.5× TBE buffer at 300 mA for 60 min. After UV crosslinking at 120 mJ/cm² in a Stratalinker UV crosslinker, the membrane was blocked with ULTRAhyb Hybridization Buffer (Invitrogen) for 30 min at 45°C and then incubated with 5′-32P labelled probe (5′-TGATAGATATTGCCCAACTACC) overnight at 45°C. The blot was washed twice with 0.1× SSC at 45°C for 15 min. The blot was exposed to Hyperfilm (GE Healthcare) for 2 days at −70°C.

To confirm the expression and size of putative sRNA, 2 μg of total RNA extracted from L. interrogans serovar Manilae were mixed together with one volume of denaturing loading buffer containing 95% formamide (Thermo Fisher), incubated at 95°C for 5 min and then placed on ice. Samples were separated by 8 M urea polyacrylamide gel (concentration ranging from 5 to 10%) in TBE buffer, along with an RNA ladder (Euromedex), for 1 h at 25 mA. The RNA integrity of samples following migration was evaluated by ethidium bromide staining (0.5 μg/mL). Gels were then transferred onto Hybond N+ membranes (Amersham) using a Criterion Blotter in TBE buffer for 1 h at 50 V. RNA molecules were crosslinked to the membranes by UV irradiation (0.51 J/cm²) and pre-hybridized with 10 mL of ULTRAhyb hybridization buffer (Thermo Fisher) for 1 h at 42°C in a rotating chamber; then, 2 μL of 10 μM 5′biotinylated oligo DNA probe (Supplementary Table 2) were added and hybridization proceeded for 14 h. Membranes were washed twice in 2X SSC and 0.1% SDS and then twice in 0.1X SSC and 0.1% SDS. Hybridized probes were visualized by incubation with horseradish peroxidase-conjugated streptavidin and chemiluminescent substrate (Thermo Fisher), followed by film exposure.

Total RNA was obtained from lymphoblastoid cell suspension cell cultures as mentioned above. Total RNA (15 μg) was separated on 1.2% formaldehyde agarose gel and transferred to Hybond-N+ nylon membrane (Amersham, United Kingdom), followed by UV crosslinking with Stratalinker 2400 (Agilent Technologies, Santa Clara, CA, United States). MECP2 cDNA probe (1431 bp), which contains a majority of exon1 and exon2, was synthesized by random primed DNA labeling kit (Roche, France) according to the manufacturer's instructions. The blots were hybridized using ULTRAhyb hybridization buffer (Applied Biosystems, Waltham, MA, United States) according to the manufacturer's instructions. In brief, the blots were hybridized with radiolabeled probes (10⁶ cpm/ml) in ULTRAhyb hybridization buffer for 2 hr at 68°C. The blots were then washed with 2× SSC, 0.05% SDS for 20 min at room temperature, followed by washing with 0.1× SSC, 0.1% SDS at 50°C for 10 min. The blots were exposed for 72 hr for analysis. For loading control, the blots were then stripped in boiling 0.2% SDS and hybridized with GAPDH probe.

An amount 10 μg of total RNAs were loaded in each well of 1.2% formaldehyde-denaturing agarose gel and after electrophoresis transferred to a charged nylon membrane overnight. After the transfer RNA was crosslinked using a UV crosslinker (Stratagene, San Diego, CA, USA) and pre-hybridized with ULTRAhyb hybridization buffer (AM8670, Applied Biosystems). Probes against mouse Malat1 transcript were amplified with the following PCR primers (forward 5′-GTTACCAGCCCAAACCTCAA-3′; reverse 5′-CTACATTCCCACCCAGCACT-3′) and labeled with [α-32P]dCTP with RadPrime DNA labeling system (Invitrogen). Probes were boiled and hybridized with membrane overnight at 42 °C. After hybridization, the membrane was washed twice with 2× SSC buffer and 0.1% SDS at room temperature and once with 0.1× SSC and 0.1% SDS at 55 °C for 30 min.